Summary Immunotherapy has transformed cancer treatment and improved clinical outcomes, but it does not cure most patients. Treatments such as anti-PD1 monoclonal antibodies and chimeric antigen receptor (CAR) T cells function by boosting the activity of cancer-specific interferon gamma-producing Th1 CD4 T cells and cytotoxic CD8 T cells (CTL). Increased effector function, however, must be balanced with the ability of anti- cancer T cells to persist long-term. A key goal of immunotherapy is to enhance effector function while maintaining T cell longevity and memory. It is now clear from work in the Rathmell lab that effector T cells utilize high rates of glycolysis while memory cells utilize mitochondrial pathways. Here I propose to test cell metabolism as a means to enhance both effector and memory T cell populations in immunotherapy. T cells radically alter their metabolism upon activation and increase glycolysis and glutamine oxidation (glutaminolysis) to support differentiation, effector function, and eventual generation of long-term memory that depend on mitochondria. Modulation of T cell glutamine metabolism may augment the efficacy of immunotherapy by enhancing both T cell effector function or memory capacity. The Rathmell Lab has shown that the metabolic program aerobic glycolysis is essential for effector T cell (Teff) function in inflammation and in tumors. Glutaminolysis complements glycolysis to fuel T cells by converting glutamine to the tricarboxylic acid cycle intermediate alpha-ketoglutarate (aKG). Using a conditional knockout of the glutaminolysis enzyme Glutaminase (GLS), which converts glutamine to glutamate, and an inhibitor of GLS that is currently in clinical trials as an anti-cancer agent, we have found that inhibition of GLS leads to a compensatory increase in glycolysis that enhances Th1 and CTL Teff function and differentiation. In addition to increasing effector function, however, I found that GLS inhibition also increases expression of inhibitory receptors, and chronic GLS deficiency ultimately suppresses T cells. In contrast, transient GLS inhibition enhanced Teff function while also priming mitochondrial metabolism for a memory-like differentiation, and led to improved T cell persistence in vivo. In this proposal, I will test the hypothesis that transient GLS inhibition can augment Teff function and maintain T cell survival and memory to boost anti-cancer immunotherapy efficacy, whereas chronic GLS inhibition will drive compensatory glycolysis, terminal Teff differentiation, and exhaustion. I will: (1) Test how transient versus chronic GLS inhibition affects CTL fate by determining differences in memory T cell formation, assessing the contribution of compensatory glycolysis to Teff phenotypes, and establishing the mitochondrial consequences of GLS inhibition; and (2) Test the effect of GLS inhibition on the immunotherapy efficacy of CD19-targeted CAR T cells and anti-PD1 treatment. These studies will demonstrate a new approach to improve anti-cancer immunotherapy and highlight a strategy of using GLS inhibition to modulate T cell metabolism and differentiation.